A genomic approach to understanding insecticide resistance in crop pests

农作物的害虫通常使用化学杀虫剂来控制。不幸的是，随着时间的推移，许多害虫已经进化出对用于控制的杀虫剂的抗性。昆虫已被证明以两种主要方式产生抗性。首先是通过杀虫剂结合的蛋白质的变化，这意味着它不再对杀虫剂的毒性作用敏感，其次是通过增加分解或结合杀虫剂并使其无效的酶的产生。在这个建议中，我们的目标是研究两种重要的作物害虫桃蚜（桃蚜）和褐飞虱（褐飞虱）的抗药性。M.在英国和欧洲，桃蚜是一系列作物的主要害虫，而在北卡罗来纳州，褐飞虱是亚洲水稻作物的主要害虫，通过直接取食和病毒传播对植物造成损害，造成很大的经济损失。这两种作物害虫已经进化出对用于其控制的许多杀虫剂的抗性，目前使用的主要化学类别是类烟碱。然而，最近有关于对这类杀虫剂产生抗药性的报告。生物化学研究表明，这种抗药性可能是由于分解杀虫剂的酶，特别是一组称为细胞色素P450单加氧酶（P450）的酶的产生增加所致。P450是一类具有许多功能的酶，包括分解毒素和昆虫，已经发现有46-143个P450基因，每个基因产生不同的酶。昆虫害虫可以通过增加它们产生的一种或多种P450酶的量来对杀虫剂产生抗性。在这个项目中，我们的目的是检查是否在M。persicae和N.从褐变到类烟碱是由P450的过量产生引起的，并确定哪些P450参与其中以及它们过量产生的原因。研究与代谢抗性有关的大基因家族并不容易，但基因组学领域（基因及其功能的研究）和新的相关技术的最新进展意味着现在更可行。本研究将利用这些新资源来鉴定目标害虫物种中的P450基因。其中包括基因组（生物体的全部DNA）许多昆虫物种的序列（包括蚜虫），表达序列标签（通过对表达基因的一端或两端进行测序产生的通常200至500个核苷酸长的小片段DNA序列）和可负担得起的高通量测序技术，（DNA的一个单位）的序列在几个小时内就可以确定。然后将使用新的分子方法研究鉴定的P450基因，该方法允许确定基因表达为RNA和蛋白质的水平。RNA干扰技术（将双链RNA引入细胞以抑制基因表达）将用于沉默P450基因，从而研究它们在抗性中的作用。最后，P450基因将被克隆并表达为蛋白质，以观察它们是否会分解或与杀虫剂结合。当参与这些作物害虫抗性的特定P450被确定后，我们将开发诊断工具来监测昆虫种群的抗性。这些是旨在减缓或防止抗药性发展的抗药性管理战略的基本要求。通过管理抗药性来延长杀虫剂的寿命至关重要，因为只有有限数量的杀虫剂可用于控制，而欧洲议会提出的新杀虫剂立法将大大减少农业中杀虫剂的可用性。该项目将与农用化学品公司的合作伙伴和抗虫害行动小组合作开展，以确保这项研究的结果能够迅速得到利用。

Insect pests of crops are often controlled using chemical insecticides. Unfortunately over time many pests have evolved resistance to the insecticides used for control. Insects have been shown to develop resistance in two main ways. Firstly by changes in the protein that the insecticide binds to which means that it is no longer as sensitive to the toxic effect of the insecticide and secondly by increased production of enzymes that break down or bind to the insecticide and render it ineffective. In this proposal we aim to study insecticide resistance in two important crop pests the peach potato aphid (Myzus persicae) and the brown planthopper (Nilaparvata lugens). M. persicae is a major pest on a range of crops in the UK and Europe and N. lugens is a major pest of rice crops in Asia, both cause damage to plants through direct feeding and the transmission of viruses resulting in high economic losses. Both of these crop pests have evolved resistance to many of the insecticides used for their control and the main chemical class currently being used is the neonicotinoids. However reports of resistance to this insecticide class have recently been described. Biochemical studies have shown that this resistance is likely to be caused by increased production of enzymes that break down the insecticide, in particular a group of enzymes called cytochrome P450 monooxgenases (P450s). P450s are a class of enzymes with many functions including the breakdown of toxins and insects have been found to have between 46-143 P450 genes, each producing a different enzyme. Insect pests can become resistant to insecticides by increasing the amount of one or more of the P450 enzymes they produce. In this project we aim to examine if resistance in M. persicae and N. lugens to neonicotinoids is caused by over-production of P450s and determine which P450s are involved and why they are over-produced. It is not easy to study the large gene families involved in metabolic resistance however recent advances in the field of genomics (the study of genes and their function) and new associated technologies means that it is now more feasible. This study will exploit these new resources to identify P450 genes in the target pest species. These include the genome (the entire DNA content of an organisms) sequences of a number of insect species (including an aphid), expressed sequence tags or ESTs (small pieces of DNA sequence usually 200 to 500 nucleotides long that are generated by sequencing either one or both ends of an expressed gene) and affordable high-throughput sequencing technologies that allow many hundreds of millions of bases (a unit of DNA) of sequence to be determined in a matter of hours. The identified P450 genes will then be studied using new molecular methods that allow determination of the levels of expression of genes into RNA and protein. The technique RNA interference (the introduction of double-stranded RNA into a cell to inhibit the expression of a gene) will be used to silence P450 genes and therefore examine their role in resistance. Finally the P450 genes will be cloned and expressed as protein to see if they break down or bind to insecticide. When the specific P450s involved in resistance in these crop pests have been identified we will develop diagnostic tools to monitor insect populations for resistance. These are an essential requirement of resistance management strategies which aim to slow or prevent the development of resistance. Prolonging the life of insecticides by managing resistance is vital as there are only a limited number of insecticides available for control and proposed new legislation on pesticides from the European Parliament will dramatically cut the availability of insecticides for use in agriculture. This project will be carried out in collaboration with partners in agrochemical companies and the Insecticide Resistance Action Group to ensure the findings of this study can be rapidly exploited.